EP0529057B1 - Proteines vehiculaires denaturees destinees a ameliorer les essais par immunosorbant lie a une enzyme - Google Patents

Proteines vehiculaires denaturees destinees a ameliorer les essais par immunosorbant lie a une enzyme Download PDF

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Publication number
EP0529057B1
EP0529057B1 EP92908459A EP92908459A EP0529057B1 EP 0529057 B1 EP0529057 B1 EP 0529057B1 EP 92908459 A EP92908459 A EP 92908459A EP 92908459 A EP92908459 A EP 92908459A EP 0529057 B1 EP0529057 B1 EP 0529057B1
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EP
European Patent Office
Prior art keywords
reactant
analysandum
enzyme
protein
immobilized
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EP92908459A
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German (de)
English (en)
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EP0529057A1 (fr
EP0529057A4 (en
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Chander P. Bahl
Eleanor Batsko
Mitchell Nelles
Gerard Garcia
David Chien
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Novartis Vaccines and Diagnostics Inc
Ortho Clinical Diagnostics Inc
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Ortho Diagnostic Systems Inc
Chiron Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV

Definitions

  • This invention pertains to enzyme linked immunosorbent assays having improved specificity. Specifically, the invention pertains to assays wherein the binding of a test sample to the vehicular protein in a recombinant fusion protein is suppressed by premixing the sample with the vehicular protein in denatured form.
  • the improved method of the present invention may be employed in a wide variety of enzyme linked immunosorbent assays to analyze a wide variety of substances.
  • ELISA enzyme linked immunosorbent assays
  • concentration of the analysandum (the substance to be analyzed) is determined by binding the analysandum with a reactant which is a protein having a specific binding affinity for the analysandum.
  • concentration of the analysandum is measured by labeling with an enzyme the reactant which has a specific binding affinity for the analysandum or a quantity of a reactant which is the same as the analysandum.
  • Enzyme labels are generally very sensitive labels because enzymes can amplify weak concentration signals through catalysis reactions.
  • Enzyme linked immunosorbent assays are classified as either homogeneous assays or heterogeneous assays.
  • a homogeneous assay the enzyme activity of the assay solution can be measured without separating the antibody bound antigens from the unbound antigens because the enzyme activity of the antibody bound antigens is significantly different from that of the unbound antigens.
  • a heterogeneous assay the enzyme activity of the assay solution is measured after the antibody bound antigens are separated, generally by immobilization, from the unbound antigens.
  • a homogeneous assay is easier to conduct and to automate but a heterogeneous assay is more sensitive.
  • a known amount of enzyme-labeled antigen competes with test sample antigen for a known limited amount of antibody to form an enzyme-labeled antigen/antibody complex.
  • the presence of the antibody in the enzyme-labeled antigen/antibody complex causes the complex to have very little enzyme activity because of steric hindrance or allosteric inhibition.
  • the enzyme activity of the assay solution is directly proportional to the amount of test sample antigen.
  • a competitive homogeneous enzyme linked immunosorbent assay is schematically set out below, wherein An is an antigen, Ab is an antibody, and Ez is an enzyme.
  • homogeneous enzyme linked immunosorbent assays are comparable to those of other immunological methods, such as radioimmunoassay, and other non-immunological methods, such as gas chromatography, high pressure liquid chromatography, and thin layer chromatography.
  • Other types of homogeneous enzyme linked immunosorbent assays are known such as assays using enzyme modulators, assays using enzyme prosthetic groups, assays using fluorogenic enzyme substrates, assays based on antibody induced restriction on the conformation of apoenzyme labeled ligand, assays using enzyme channeling, assays using liposome-entrapped enzymes, and assays using reagent strip format.
  • heterogeneous enzyme linked immunosorbent assays are essentially the same as those for radioimmunoassays.
  • a competitive heterogeneous enzyme linked immunosorbent assay a known amount of soluble enzyme-labeled antigen competes with test sample antigen for a known limited amount of immobilized antibody. After reaction, the immobilized antibody phase containing the bound enzyme-labeled antigen is separated from the soluble phase. The enzyme activity of the enzyme-labeled antigen/immobilized antibody phase is inversely proportional to the amount of test sample antigen.
  • a competitive heterogeneous enzyme linked immunosorbent assay is schematically set out below, wherein An is an antigen, Ab is an antibody, Ez is an enzyme, and phase is an immobilized phase.
  • an inhibition heterogeneous enzyme linked immunosorbent assay a known amount of immobilized antigen competes with test sample antigen for a known limited amount of soluble enzyme-labeled antibody. After reaction, the immobilized antigen phase containing the bound enzyme-labeled antibody is separated from the soluble phase. The enzyme activity of the enzyme-labeled antibody/immobilized antigen phase is inversely proportional to the amount of test sample antigen.
  • Sandwich heterogeneous enzyme linked immunosorbent assays are used to analyze for antigens which have multiple epitopes (the site or determinant on an antigen which attaches to an antibody) or antibodies which have multiple paratopes (the site on an antibody which attaches to an antigen).
  • a test sample antigen which has multiple epitopes, is reacted with an excess of immobilized antibody-1.
  • an excess or known amount of enzyme-labeled antibody-2 is added to the immobilized phase to further react with the antigen.
  • the enzyme activity of the enzyme-labeled antibody-2/antigen/immobilized antibody-1 phase is directly proportional to the amount of test sample antigen.
  • Indirect sandwich heterogeneous enzyme linked immunosorbent assays are used to measure serum levels of specific antibodies produced in response to a pathogen.
  • a test sample antibody-1 which has multiple paratopes, is reacted with an excess of immobilized antigen.
  • an excess or known amount of enzyme-labeled antispecies immunoglobulin antibody-2 is added to the immobilized phase to further react with antibody-1.
  • the enzyme activity of the enzyme-labeled antibody-2/antibody-1/immobilized antigen phase is directly proportional to the amount of antibody in the test sample.
  • the first immunochemical reaction can be carried out in a volume larger than is desirable for the second reaction such as when the analysandum is present in a low concentration.
  • the biological fluid in the first reaction may contain substances which would adversely effect the second immunochemical reaction, the enzyme present in the conjugate, or the subsequent enzymatic assay reaction.
  • Enzyme linked immunosorbent assays are reviewed in detail in Enzyme Mediated Immunoassays, Ngo and Lenhoff (Editors), "Enzyme Mediated Immunoassay: An Overview", pp. 3-32, Plenum Press, New York, N.Y. (1985).
  • United States patent Re. no. 31,006, issued to Schuurs et al. describes a process for the demonstration and determination of a component of the reaction between specific binding proteins.
  • the process comprises reacting the component to be determined with its binding partner in insolubilized form, separating the solid phase of the reaction mixture from the liquid phase, reacting the solid phase with a coupling product obtained by binding a substance capable of reacting specifically with one of the reaction components to an enzyme, and determining the enzyme activity of the liquid or the solid phase of the reaction mixture obtained, which is a measure of the quantity of the substance to be determined.
  • the present invention pertains to a homogeneous enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the present invention pertains to a heterogeneous enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the present invention pertains to a heterogeneous sandwich enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the present invention pertains to enzyme linked immunosorbent assays for analyzing a test sample or analysandum which is a reactant in a reaction of binding counterparts.
  • the binding counterparts comprise a bindable substance such as an antigen or a hapten and a binding substance such as an antibody or a specific binding protein.
  • a first reactant which is the binding counterpart of the analysandum, is provided as a recombinant fusion protein.
  • a recombinant fusion protein comprises a first protein having the amino acid sequence and immunological reactivity of the binding counterpart and a second protein which is a vehicular or carrier protein. The first and second proteins are fused to each other.
  • a second reactant which is an enzyme-labeled reactant comprising a reactant which is the same as the analysandum coupled to an enzyme.
  • a diluent solution of the analysandum is prepared by forming a solution of the analysandum with an effective amount of denatured vehicular protein to suppress binding of the analysandum to the vehicular protein in the recombinant fusion protein.
  • the diluent solution of analysandum and the enzyme-labeled second reactant are then competitively reacted with the recombinant fusion protein first reactant to assay for the analysandum.
  • Applicants have found that a certain amount of non-specific binding often occurs between a test sample and the vehicular protein portion of a recombinant fusion protein to provide a false positive response and thereby decrease the specificity of the assay. Addition of native vehicular protein to the test sample solution as a competitive inhibitor does not suppress this non-specific binding. Applicants have discovered that by adding denatured vehicular protein to the test sample solution, the non-specific binding between the test sample and the vehicular protein portion of the recombinant fusion protein is suppressed.
  • the improved method of the present invention may be used to increase the specificity of a wide variety of enzyme linked immunosorbent assays to analyze a wide variety of substances.
  • the enzyme linked immunosorbent assays within the scope of the present invention include homogeneous assays and heterogeneous assays.
  • the homogeneous enzyme linked immunosorbent assays may be assays using enzyme modulators, assays using enzyme prosthetic groups, assays using fluorogenic enzyme substrates, assays based on antibody induced restriction on the conformation of apoenzyme labeled ligand, assays using enzyme channeling, assays using liposome-entrapped enzymes, and assays using reagent strip format.
  • the heterogeneous enzyme linked immunosorbent assays may be competitive assays, inhibition assays, direct and indirect sandwich assays, immunoenzymometric assays, tagged enzyme ligand conjugate assays, steric hindrance assays, isoenzyme assays, and amplified enzyme label assays.
  • the immunoassay is a heterogeneous assay such as a competitive assay, an inhibition assay, a direct sandwich assay, and an indirect sandwich assay.
  • the immunoassay is a heterogeneous direct sandwich assay or a heterogeneous indirect sandwich assay.
  • the heterogeneous immunoassay is an indirect sandwich assay.
  • the analysandum or substance to be analyzed in the enzyme linked immunosorbent assay of the present invention is a reactant in a reaction between binding counterparts.
  • the binding counterparts are proteins which have a specific binding affinity for each other.
  • One binding counterpart is a bindable substance which is selected from the group consisting of an antigen and a hapten.
  • the preferred bindable substance is an antigen.
  • the other binding counterpart is a binding substance which is selected from the group consisting of an antibody and a specific binding protein.
  • the preferred binding substance is an antibody.
  • Antigens are substances which are capable under appropriate conditions of inducing the formation of antibodies and of reacting specifically in some detectable manner with the antibodies so induced.
  • Antigens may be soluble substances, such as toxins and foreign proteins, or particulate substances, such as bacteria or tissue cells.
  • antigens are high molecular weight substances such as simple and conjugated proteins and carbohydrates.
  • the bindable substance may also be a low molecular weight substance such as a hapten.
  • Haptens are specific protein-free substances which have a chemical configuration which can interact with specific binding groups on an antibody but which, unlike antigenic determinants, does not itself elicit the formation of a detectable amount of antibody.
  • haptens When haptens are coupled with a carrier protein to form a conjugate, the hapten can elicit an immune response.
  • humoral immunity antibody specificity is directed primarily at the hapten.
  • cell mediated immunity antibody specificity is directed at both the hapten and the carrier protein.
  • the bindable substances in the present invention are substances from natural sources or may be substances prepared by synthetic or recombinant means.
  • the bindable substance is an antigen selected from the group consisting of recombinant proteins and synthetic peptides.
  • the antigen is selected from the group consisting of hepatitus C virus (HCV) and human immunedeficiency virus (HIV).
  • Antibodies are immunoglobulin molecules which have a specific amino acid sequence which permit it to interact only with the antigen which induced its synthesis in lymphoid tissue or with an antigen closely related to that antigen.
  • Immunoglobulins are proteins made up of two light chains and two heavy chains.
  • the binding substance may also be a specific binding protein such as an unattached receptor protein or a transport protein.
  • Receptor proteins include proteins which remain attached to cells such as antibodies and unattached proteins which are released to blood serum and retain their specific binding affinity.
  • Transport proteins are proteins that move substances in and out of cells and across epithelial layers in biological systems.
  • the binding substances may be substances from natural sources or may be substances prepared by synthetic or recombinant means.
  • the bindable substance is an antibody selected from the group consisting of recombinant proteins and synthetic peptides.
  • the antibody is selected from the group consisting of hepatitus C virus antibody and human immunedeficiency virus antibody.
  • the medium in which the analysandum occurs may be any biological fluid such as serum, plasma, urine, cell culture medium, or synovial fluid.
  • the analysandum may be analyzed directly in the biological fluid, may be extracted from the fluid and concentrated, or may be diluted to decrease the influence of competing reactions.
  • the pH value of the biological fluid or test sample may be adjusted to a pH value to optimize the immunochemical reaction.
  • the pH value of the test sample may be adjusted to a value from about 5 to about 9.5, preferably from about 8.5 to about 9.5, and more preferably about 9.
  • the pH value of the test sample may be adjusted by adding to the fluid a small amount of a concentrated buffer solution or a dry buffer salt.
  • Typical buffer systems include borate buffers, phosphate buffers, citrate buffers, tris(hydroxymethyl)aminomethane buffers, imidazole buffers, and carbonate buffers.
  • the reactant which is the binding counterpart of the test sample in the present invention is provided as a recombinant fusion protein.
  • the recombinant fusion protein comprises a first protein having the immunological reactivity of the binding counterpart and a second protein which is a vehicular protein fused to the first protein.
  • the vehicular protein in the recombinant fusion protein may be any protein suitable for fusing to the first protein to enhance the yield, stability, and ease of isolation of the recombinant fusion protein and to improve the assay sensitivity.
  • Recombinant fusion proteins and methods for preparing such proteins are discussed in detail in European patent application no. 318,216, published 18 November 1989, and European patent application no. 388,232, published 19 September 1989.
  • the binding of a test sample to the vehicular protein in a recombinant fusion protein is suppressed by premixing the test sample with a diluent solution of the vehicular protein in denatured form.
  • the vehicular protein in the diluent solution will be the same vehicular protein, or the same type of vehicular protein present, in the recombinant fusion protein.
  • Nonlimiting examples of vehicular proteins include superoxide dismutase, beta -galactosidase, and lactamase.
  • the vehicular protein is selected from the group consisting of superoxide dismutase and beta -galactosidase, and in a more preferred embodiment, the vehicular protein is superoxide dismutase.
  • the amount of vehicular protein in the diluent solution is an effective amount to suppress binding of the analysandum to the vehicular protein in the recombinant fusion protein.
  • An effective amount of vehicular protein is a matter of preference subject to such factors as the type and binding affinity of vehicular protein employed, the amount and type of test sample being assayed, and the level of specificity desired.
  • the exact amount of vehicular protein may be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation.
  • the vehicular protein will be present in the diluent solution in an amount from about 30 ⁇ g/ml to about 300 ⁇ g/ml, preferably from about 100 ⁇ g/ml to about 200 ⁇ g/ml, and more preferably about 100 ⁇ g/ml, by weight of the diluent solution.
  • the pH value of the diluent solution will be adjusted to optimize the immunochemical reaction.
  • the pH value of the diluent solution may be adjusted to a value from about 5 to about 9.5, preferably from about 6 to about 8, and more preferably about 7.3.
  • Typical buffer systems which can be employed to adjust the pH value of the diluent solution have been described above.
  • the diluent solution also may be formulated with conventional ingredients which offer a variety of properties to suit particular applications.
  • ingredients include preservatives, salts to increase the ionic strength of the solution, and various proteins to reduce non-specific binding of the test sample such as detergents, yeast extract, casein, and bovine serum albumin.
  • the vehicular protein in the diluent solution may be denatured in any conventional manner.
  • the vehicular protein may be denatured using denaturing agents (e.g., chaotropic agents) or may be denatured with reduction, heat, or with a detergent.
  • denaturing agents e.g., chaotropic agents
  • Methods for denaturing the vehicular proteins are well known in the art.
  • the premix of test sample and diluent solution of denatured vehicular protein may be employed in any homogeneous or heterogeneous enzyme linked immunosorbent assay to analyze a wide variety of substances.
  • the premixture of test sample in the diluent solution competes with a known amount of enzyme-labeled reactant, which is the same as the test sample, for a known limited amount of binding counterpart, which is provided as a recombinant fusion protein.
  • the invention is directed at an enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the premixture of test sample in the diluent solution reacts with an immobilized reactant, which is provided as a recombinant fusion protein.
  • the invention is directed at enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the immobilized reactant in the heterogeneous assay is preparing by mixing the reactant with an aqueous solution having an ionic strength value from about 0.05 to about 1.0 and contacting the mixture with a solid phase to immobilize the reactant on the solid phase.
  • the pH value of the reactant solution may be adjusted to optimize the immunochemical reaction.
  • the ionic strength value of the reactant mixture is that value sufficiently high to minimize ionic binding of the reactant to the solid phase and thereby decrease protein leaching during later stages of the assay.
  • the ionic strength value of the reactant mixture must also be sufficiently low so as not to adversely affect the solubility of the protein or otherwise interfere with the binding of the reactant to the solid phase.
  • the exact ionic strength value is subject to such factors as the type of reactant to be immobilized and the type of solid phase employed to immobilize the reactant. Thus the ionic strength value may be varied in order to obtain the result desired in the final assay and such variations are within the capabilities of those skilled in the art without the need for undue experimentation.
  • the ionic strength value of the mixture of reactant to be immobilized is from about 0.05 to about 1.0, preferably from about 0.1 to about 1, and more preferably from about 0.25 to about 1. Ionic strength values from about 2 to about 5 tend to have a negative effect on the sensitivity of the assay.
  • the types of salts useful in the present invention are salts which are capable of increasing the ionic strength value of the reactant mixture without interfering with the binding of the reactant to the solid phase or otherwise adversely affecting the sensitivity of the assay.
  • the exact type of salt employed is a matter of preference subject to such factors as the type and concentration of reactant being immobilized and the type of solid phase being employed.
  • Suitable salts that may be employed in the present invention may be selected from the group consisting of alkali metal salts, alkaline earth metal salts, and the like, and mixtures thereof.
  • the salt may be selected from the group consisting of sodium chloride, potassium chloride, and mixtures thereof.
  • the salt is sodium chloride. Salts may be added in solid form or solution form.
  • the solid phase in which the reactant is bound and immobilized may be any commercially available solid phase generally used for this purpose.
  • Typical solid phases include a microwell as well as solid phase particles in a tube or container.
  • the solid phase will be made of a material which will bind and immobilize the reactant by a combination of ionic and hydrophobic interaction forces.
  • Suitable materials for solid phases are polymeric materials which have hydrophobic and hydrophilic sites such as those polymers selected from the group consisting of polystyrene, sulfonated polystyrene, irradiated (modified) sulfonated polystyrene, latex, and mixtures thereof.
  • the solid phase is sulfonated polystyrene.
  • the aqueous reactant mixture is contacted with the solid phase for a time sufficient for the reactant to bind and be immobilized on the solid phase.
  • the aqueous reactant mixture will be contacted with the solid phase for from about 1 to 6 hours at ambient temperature.
  • the aqueous mixture is separated from the solid phase by any convenient means such as aspiration or decantation to prepare the immobilized reactant.
  • the solid phase may optionally be rinsed or washed with a detergent to remove any non-immobilized or soluble reactant.
  • the immobilized reactant in the solid phase may optionally be treated with an aqueous solution of an excess of a protein which is a nonreactant in the reaction of binding counterparts.
  • the nonreactant is a protein which will react or saturate any remaining binding or active sites on the solid phase but will not react or interfere with subsequent immunoassay reactions. Treatment of the solid phase with a nonreactant protein helps to prevent non-specific binding of the analysandum to the solid phase. Suitable nonreactant proteins include bovine serum albumen (BSA), gelatin, and ovalvumin.
  • BSA bovine serum albumen
  • the nonreactant protein will be present in an excess amount to bind all active sites in the solid phase, such as in a concentration of about lmg to 3mg per ml of solution.
  • the aqueous solution of nonreactant is then separated from the solid phase and the solid phase is dried to prepare the immobilized reactant.
  • the assay may be a competitive assay or an inhibition assay.
  • a competitive assay a reactant which is the binding counterpart of the analysandum is immobilized in a solid phase according to the method of the present invention.
  • An enzyme conjugate is then prepared by coupling an enzyme and a reactant which has the same chemical structure as the analysandum.
  • the analysandum is then mixed with a known quantity of the enzyme conjugate and reacted with a known limited quantity of the immobilized reactant to form a reaction mixture having an immobilized phase and a liquid phase.
  • the analysandum and the enzyme conjugate compete for the limited quantity of immobilized reactant.
  • the known limited quantity of the immobilized reactant corresponds to a quantity which is not sufficient to react with all of the analysandum and the enzyme conjugate.
  • the immobilized phase and liquid phase are then separated.
  • the enzyme activity of the immobilized phase is inversely proportional to the quantity of analysandum.
  • a reactant which is the same as the analysandum is immobilized in a solid phase according to the method of the present invention.
  • An enzyme conjugate is then prepared by coupling an enzyme and a reactant which is the binding counterpart of the analysandum.
  • the analysandum is then mixed with a known quantity of the immobilized reactant and reacted with a known limited quantity of the enzyme conjugate to form a reaction mixture having an immobilized phase and a liquid phase.
  • the analysandum and the immobilized reactant compete for the limited quantity of enzyme conjugate.
  • the known limited quantity of the enzyme conjugate corresponds to a quantity which is not sufficient to react with all of the analysandum and the immobilized reactant.
  • the immobilized phase and liquid phase are then separated.
  • the enzyme activity of the immobilized phase is inversely proportional to the quantity of analysandum.
  • the enzyme conjugate is a coupling product of a reactant in the enzyme linked immunosorbent assay and an enzyme.
  • the enzyme may be any enzyme which does not interfere or is not affected by the immunochemical chemical reaction.
  • the exact choice of enzyme is subject to such factors as the ease of the synthesis of the enzyme conjugate, the specific binding activity of the enzyme (a high conversion rate raises the specificity of the assay), and the simplicity of the enzyme assay.
  • Suitable enzymes which may be use in the enzyme conjugate may be selected from the group consisting of catalase, peroxidases such as horse radish peroxidase (HRP), beta-glucuronidase, beta-B-glucosidase, beta-D-galactosidase, urease, glucose-oxidase, galactose-oxidase, and alkaline phosphatase.
  • HRP horse radish peroxidase
  • beta-glucuronidase beta-B-glucosidase
  • beta-D-galactosidase urease
  • glucose-oxidase galactose-oxidase
  • alkaline phosphatase alkaline phosphatase
  • the enzyme is horse radish peroxidase.
  • the preparation of the enzyme conjugate may be carried out by any conventional method subject to such factors as the properties of the particular enzyme and specific binding protein.
  • the enzyme conjugate may be prepared with reagents such as carbodiimides, diisocyanates, glutaric aldehyde, and bis-diazobenzidine.
  • the preparation of the enzyme conjugate should not significantly affect the binding properties of the protein or the activity of the enzyme.
  • the activity of the enzyme conjugate in the immobilized phase may be measured by reacting the immobilized enzyme conjugate with an enzyme substrate and measuring the conversion rate or activity of the enzyme.
  • Suitable enzyme substrates include 1,2-phenylenediamine ( ortho -phenylenediamine), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 3,3',5,5'-tetramethylbenzidine (TME).
  • the activity of the enzyme may be measured by any conventional method such as by spectrophotometry, fluorimetry, or colorimetry.
  • the invention is directed at a sandwich enzyme linked immunosorbent assay for analyzing an analysandum which is a reactant in a reaction between binding counterparts wherein the counterparts comprise a bindable substance and a binding substance, which comprises the steps of:
  • the sandwich assay may be a direct sandwich assay or an indirect sandwich assay.
  • a reactant which is a first antibody to the antigen analysandum, and which is provided as a recombinant fusion protein, is immobilized in a solid phase.
  • the analysandum is then reacted with an excess quantity of the immobilized reactant to form a first reaction mixture having a first immobilized phase and a first liquid phase.
  • the first immobilized phase is separated from the first liquid phase.
  • An enzyme conjugate is prepared by coupling an enzyme and a second antibody which has a specific affinity for the antigen analysandum.
  • the first immobilized phase containing the antigen analysandum bound to the immobilized antibody reactant is then reacted with a known quantity of the enzyme conjugate to form a second reaction mixture having a second immobilized phase and a second liquid phase.
  • the second immobilized phase is then separated from the second liquid phase.
  • the enzyme activity of the immobilized phase is directly proportional to the quantity of analysandum.
  • a reactant which is a first antigen to the antibody analysandum, and which is provided as a recombinant fusion protein is immobilized in a solid phase.
  • the analysandum is then reacted with an excess quantity of the immobilized reactant to form a first reaction mixture having a first immobilized phase and a first liquid phase.
  • the first immobilized phase is separated from the first liquid phase.
  • An enzyme conjugate is prepared by coupling an enzyme and a second binding protein, such as an antispecies antibody, having a specific binding affinity for the antibody analysandum.
  • the first immobilized phase containing the antibody analysandum bound to the immobilized antigen reactant is then reacted with a known quantity of the enzyme conjugate to form a second reaction mixture having a second immobilized phase and a second liquid phase.
  • the second immobilized phase is then separated from the second liquid phase.
  • the enzyme activity of the immobilized phase is directly proportional to the quantity of analysandum.
  • the enzyme linked immunosorbent assay is an indirect sandwich assay.
  • the known quantity of the enzyme-labeled reactant in the sandwich assay is a predetermined quantity which will minimize nonspecific binding of the enzyme-labeled reactant to the solid phase (negative signal) and maximize the reaction of the enzyme-labeled reactant with the first immobilized phase containing the analysandum (positive signal).
  • the assay range in an enzyme linked immunosorbent assay is very wide because the analysandum may be present in high or low amounts.
  • the known quantity of the enzyme-labeled reactant is an intermediate quantity which will minimize nonspecific binding and maximize the reaction of the enzyme-labeled reactant with the analysandum. This known quantity of the enzyme-labeled reactant may be an excess quantity when the analysandum is present in low amounts and may not be an excess quantity when the analysandum is present in high amounts.
  • the second antibody which has a specific binding affinity for the antigen analysandum or the second binding protein which has a specific binding affinity for the antibody analysandum may be the same protein as the first binding counterpart in the sandwich assay or may be a third reactant which is a different protein.
  • the third reactant may be an antispecies antibody which is an antibody which reacts with an antibody from another species, such as a mouse antispecies antibody which reacts with a human antibody.
  • the second specific binding protein in the indirect assay is a third reactant which is an antispecies antibody.
  • This example demonstrates the improved method of the present invention in an indirect sandwich enzyme linked immunosorbent assay. Specifically, the binding of a test sample (antibodies to hepatitis C virus) to the vehicular protein (superoxide dismutase) in a recombinant fusion protein was suppressed by premixing the test sample with the vehicular protein in denatured form.
  • Immobilized antigen reactant solutions or antigen coating solutions were made with the following recombinant superoxide dismutase (SOD) fusion proteins.
  • SOD superoxide dismutase
  • the antigens C-100-3, C-22, and C-200 are recombinant superoxide dismutase fusion proteins for hepatitis C virus expressed in yeast.
  • the fusion protein C-100-3 is described in published European patent application no. 388,232.
  • the preparation and purification of fusion proteins C-22 and C-200 are carried out as described on pages 29-33 of published European patent application no. 388,232.
  • the amino acid sequence of fusion protein C-100-3 is amino acids numbers 1569-1931
  • the amino acid sequence of fusion protein C-22 is amino acid numbers 2-120
  • the amino acid sequence of fusion protein C-200 is amino acids numbers 1192-1930.
  • Immulon-1 microwells sulfonated polystyrene, Dynatech, Chantilly, Virginia
  • PBS phosphate buffered saline
  • a sample diluent solution in phosphate buffered saline (pH 7.3) was prepared having the following components.
  • Denatured Superoxide Dismutase conveniently (100 ⁇ g/ml-200 ⁇ g/ml) preferably (100 ⁇ g/ml) 30ug/ml-300ug/ml
  • the superoxide dismutase in the sample diluent solution was denatured by mixing a solution of SOD (70mg/ml) with a solution of urea (420mg/ml) and dithithreitol (1.54mg/ml). The resulting solution was then heated to about 60-65° C. for about two hours.
  • Wash buffer solution is available commercially under the tradename Ortho 20X Wash Buffer Concentrate from Ortho Diagnostics Systems Inc. Raritan, New Jersey.
  • the enzyme conjugate solution which is horseradish peroxidase conjugated to anti-human IpG (monoclonal) in phosphate buffered saline (pH 7.4) with stabilizing proteins, is available commercially from Ortho Diagnostics Systems Inc. Raritan, New Jersey.
  • the enzyme substrate which is ortho-phenylenediamine (OPD) in water with hydrogen peroxide, is available commercially from Ortho Diagnostics Systems Inc. Raritan, New Jersey as tablets mixed with substrate buffer (6ml).
  • the enzyme linked immunosorbent assay was carried out as follows. A quantity of 20 ⁇ l of analysandum or sample (serum or plasma) was diluted in 200ul of sample diluent and added to an antigen coated microwell as set out above. The microwell was incubated for a period of about one hour at 37° C. The sample solution was then removed from the well by aspiration and the well was rinsed five times with wash buffer.
  • a quantity of 200 ⁇ l of enzyme conjugate solution was added to the microwell and the well was incubated for a period of one hour at 37°C.
  • the conjugate solution was then removed from the well by aspiration and the well was washed five times with wash buffer.
  • a quantity of 200 ⁇ l of enzyme substrate solution was added to the microwell and the well was incubated for a period of 30 minutes at room temperature.
  • a quantity of 50 ⁇ l of 4N sulfuric acid solution was then added to the well and the optical density (OD) of the well was read at 490 NM. Reactivity was determined at a cutoff of 0.400 + mean of negative control optical density. Samples with an optical density at or above the cutoff were considered reactive for hepatitis C virus antibody.
  • Table 1 shows that samples 2, 4, 10, and 11 generated false positive signals when the test samples were not treated with denatured superoxide dismutase.
  • the number of false positive signals was not suppressed in the control samples without added superoxide dismutase or the samples containing native superoxide dismutase.

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Claims (20)

  1. Dosage par immunosorption à liaison enzymatique (E.L.I.S.A.) pour analyser un analysandum qui est un réactif dans une réaction entre des contreparties de liaison dans lequel les contreparties comprennent une substance susceptible de se lier et d'une substance liante, qui comprend les étapes consistant à :
    (a) se munir d'un premier réactif qui est la contrepartie de liaison de l'analysandum en tant que protéine de fusion recombinante, dans lequel la protéine de fusion comprend une première protéine ayant la réactivité immunologique du premier réactif et une seconde protéine qui est une protéine vectrice fusionnée à la première protéine ;
    (b) se munir d'un second réactif qui est un réactif marqué par une enzyme dans lequel le réactif marqué par une enzyme comprend un réactif qui est le même que l'analysandum couplé à une enzyme ;
    (c) préparer une solution diluée de l'analysandum en formant une solution de l'analysandum avec une quantité efficace de protéine vectrice dénaturée pour supprimer la liaison de l'analysandum à la protéine vectrice dans la protéine de fusion recombinante ;
    (d) faire réagir la solution diluée d'analysandum de l'étape (c) avec le réactif constitué par la protéine de fusion recombinante de l'étape (a) et le réactif marqué par une enzyme de l'étape (b) pour former un mélange réactionnel, dans lequel le réactif marqué par une enzyme est présent en quantité connue et le réactif constitué par la protéine de fusion recombinante est présent en quantité connue, insuffisante pour réagir avec la quantité totale d'analysandum et de réactif marqué par une enzyme ; et
    (e) analyser l'activité enzymatique du mélange réactionnel qui est directement proportionnelle à la quantité d'analysandum présente dans le mélange réactionnel.
  2. Dosage selon la revendication 1, dans lequel la substance susceptible de se lier est un antigène choisi dans le groupe constitué par le virus de l'hépatite C et le virus HIV.
  3. Dosage selon la revendication 1 ou 2, dans lequel la protéine vectrice dans l'étape (a) est choisie dans le groupe constitué par la superoxyde dismutase, la β-galactosidase et la lactamase.
  4. Dosage selon la revendication 3, dans lequel la protéine vectrice dans l'étape (a) est la superoxyde dismutase.
  5. Dosage par immunosorption à liaison enzymatique (E.L.I.S.A.) pour analyser un analysandum qui est un réactif dans une réaction entre des contreparties de liaison dans lequel les contreparties comprennent une substance susceptible de se lier et une substance liante, qui comprend les étapes consistant à:
    (a) se munir d'un premier réactif qui est la contrepartie de liaison de l'analysandum en tant que protéine de fusion recombinante, dans lequel la protéine de fusion comprend une première protéine ayant la réactivité immunologique du premier réactif et une seconde protéine qui est une protéine vectrice fusionnée à la première protéine ;
    (b) immobiliser le réactif constitué de la protéine de fusion recombinante de l'étape (a) en mettant en contact une solution aqueuse de la protéine de fusion recombinante avec une phase solide pour immobiliser la protéine de fusion sur la phase solide ;
    (c) séparer la solution aqueuse de la phase solide dans l'étape (b) pour préparer un réactif immobilisé ;
    (d) se munir d'un second réactif qui est un réactif marqué par une enzyme dans lequel le réactif marqué par une enzyme comprend un réactif qui est le même que l'analysandum couplé à une enzyme ;
    (e) préparer une solution dans un diluant de l'analysandum en formant une solution de l'analysandum avec une quantité efficace de protéine vectrice dénaturée pour supprimer la liaison de l'analysandum à la protéine vectrice dans la protéine de fusion recombinante ;
    (f) faire réagir la solution dans le diluant d'analysandum de l'étape (e) avec le réactif constitué par la protéine de fusion recombinante immobilisée de l'étape (c) et le réactif marqué par une enzyme de l'étape (d) pour former un mélange réactionnel, dans lequel le réactif marqué par une enzyme est présent en quantité connue et le réactif constitué par la protéine de fusion recombinante est présent en quantité connue, insuffisante pour réagir avec la quantité totale d'analysandum et de réactif marqué par une enzyme ;
    (g) séparer la phase immobilisée et la phase liquide ; et
    (h) analyser l'activité enzymatique de la phase immobilisée qui est inversement proportionnelle à la quantité d'analysandum présente dans le mélange réactionnel.
  6. Dosage selon la revendication 5, dans lequel la substance susceptible de se lier est un antigène choisi dans le groupe constitué par le virus de l'hépatite C et le virus HIV.
  7. Dosage selon la revendication 5 ou 6, dans lequel la protéine vectrice dans l'étape (a) est choisie dans le groupe constitué par la superoxyde dismutase, la β-galactosidase et la lactamase.
  8. Dosage selon l'une quelconque des revendications 5 à 7, dans lequel la solution aqueuse dans l'étape (b) possède une force ionique d'environ 0,05 à environ 1,0.
  9. Dosage selon l'une quelconque des revendications 5 à 8, dans lequel la phase solide est faite d'un polymère choisi dans le groupe constitué par le polystyrène, le polystyrène sulfoné, le polystyrène sulfoné irradié et le latex.
  10. Dosage selon l'une quelconque des revendications 5 à 9, comprenant par ailleurs les étapes consistant à (i) mettre en contact le réactif immobilisé dans l'étape (b) avec une solution aqueuse d'une protéine qui est non réactive dans la réaction pour enrober les sites actifs sur la phase solide et (ii) séparer la solution aqueuse du réactif immobilisé.
  11. Dosage par immunosorption à liaison enzymatique (E.L.I.S.A.) pour analyser un analysandum qui est un réactif dans une réaction entre des contreparties de liaison dans lequel les contreparties comprennent une substance susceptible de se lier et une substance liante, qui comprend les étapes consistant à:
    (a) se munir d'un premier réactif qui est la contrepartie de liaison de l'analysandum en tant que protéine de fusion recombinante, dans lequel la protéine de fusion comprend une première protéine ayant la réactivité immunologique du premier réactif et une seconde protéine qui est une protéine vectrice fusionnée à la première protéine ;
    (b) immobiliser le réactif constitué de la protéine de fusion recombinante de l'étape (a) en mettant en contact une solution aqueuse de la protéine de fusion recombinante avec une phase solide pour immobiliser la protéine de fusion sur la phase solide ;
    (c) séparer la solution aqueuse de la phase solide dans l'étape (b) pour préparer un réactif immobilisé ;
    (d) préparer une solution diluée de l'analysandum en formant une solution de l'analysandum avec une quantité efficace de protéine vectrice dénaturée pour supprimer la liaison de l'analysandum à la protéine vectrice dans la protéine de fusion recombinante ;
    (e) faire réagir la solution diluée d'analysandum de l'étape (d) avec le réactif constitué par la protéine de fusion recombinante immobilisée de l'étape (c) pour former un premier mélange réactionnel comportant une première phase immobilisée et une première phase liquide, dans lequel le réactif immobilisé est présent en quantité suffisante pour réagir avec la totalité de l'analysandum ;
    (f) séparer la première phase immobilisée et la première phase liquide dans l'étape (e);
    (g) se munir d'un second réactif qui est un réactif marqué par une enzyme dans lequel le réactif marqué par une enzyme comprend un réactif qui est une seconde contrepartie de liaison de l'analysandum couplé à une enzyme ;
    (h) faire réagir le réactif marqué par une enzyme de l'étape (g) avec la première phase immobilisée de l'étape (f) pour former un second mélange réactionnel comportant une seconde phase immobilisée et une seconde phase liquide, dans lequel le réactif marqué par une enzyme est présent en quantité connue ;
    (i) séparer la seconde phase immobilisée et la seconde phase liquide dans l'étape (h) ; et
    (j) analyser l'activité enzymatique de la seconde phase de produit immobilisée dans l'étape (i), qui est directement proportionnelle à la quantité d'analysandum présente dans le mélange réactionnel.
  12. Dosage selon la revendication 11, dans lequel l'analysandum est une substance liante choisie dans le groupe constitué par un anticorps et une protéine liante spécifique.
  13. Dosage selon la revendication 12, dans lequel la substance liante est un anticorps choisi dans le groupe constitué par l'anticorps du virus de l'hépatite C et l'anticorps du virus HIV.
  14. Dosage selon la revendication 11, dans lequel l'analysandum est une substance susceptible de se lier choisie dans le groupe constitué par un antigène et un haptène.
  15. Dosage selon l'une quelconque des revendications 11 à 14, dans lequel la protéine vectrice dans l'étape (a) est choisie dans le groupe constitué par la superoxyde dismutase, la β-galactosidase et la lactamase.
  16. Dosage selon l'une quelconque des revendications 11 à 15, dans lequel la solution aqueuse dans l'étape (b) possède une force ionique d'environ 0,05 à environ 1.
  17. Dosage selon l'une quelconque des revendications 11 à 16, dans lequel la phase solide est faite d'un polymère choisi dans le groupe constitué par le polystyrène, le polystyrène sulfoné, le polystyrène sulfoné irradié et le latex.
  18. Dosage selon l'une quelconque des revendications 11 à 17, comprenant par ailleurs les étapes consistant à (i) mettre en contact le réactif immobilisé dans l'étape (b) avec une solution aqueuse d'une protéine qui est non réactive dans la réaction pour enrober les sites actifs sur la phase solide et (ii) séparer la solution aqueuse du réactif immobilisé.
  19. Dosage selon l'une quelconque des revendications 11 à 18, dans lequel la seconde contrepartie de liaison de l'analysandum dans l'étape (g) est un troisième réactif différent du réactif dans l'étape (a) et est un anticorps anti-espèce.
  20. Dosage selon l'une quelconque des revendications 11 à 18, dans lequel la seconde contrepartie de liaison de l'analysandum dans l'étape (g) est la même que le réactif dans l'étape (a).
EP92908459A 1991-03-06 1992-03-05 Proteines vehiculaires denaturees destinees a ameliorer les essais par immunosorbant lie a une enzyme Expired - Lifetime EP0529057B1 (fr)

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US66503691A 1991-03-06 1991-03-06
US665036 1991-03-06
PCT/US1992/001611 WO1992015881A1 (fr) 1991-03-06 1992-03-05 Proteines vehiculaires denaturees destinees a ameliorer les essais par immunosorbant lie a une enzyme

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US6511812B1 (en) * 1994-05-09 2003-01-28 Abbott Laboratories Method and test kit for use in improving immunoassay specificity
WO1995030902A1 (fr) * 1994-05-09 1995-11-16 Abbott Laboratories Procede et reactifs permettant d'ameliorer la specificite d'un dosage immunologique
US8613943B2 (en) 2009-01-23 2013-12-24 Royal College Of Surgeons In Ireland Process for producing a multi-layered scaffold suitable for osteochondral repair

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US4407943A (en) * 1976-12-16 1983-10-04 Millipore Corporation Immobilized antibody or antigen for immunoassay
US4256724A (en) * 1978-02-21 1981-03-17 Becton, Dickinson And Company Method for non-covalent coating of antibodies on solid substrates
US4820642A (en) * 1983-04-04 1989-04-11 The Regents Of The University Of California Amplified expression vector
DE3314812A1 (de) * 1983-04-23 1984-10-25 Battelle-Institut E.V., 6000 Frankfurt Mittel zum antigen/antikoerper-nachweis
JPS60256057A (ja) * 1984-06-01 1985-12-17 Dai Ichi Pure Chem Co Ltd 免疫学的測定法
US4745182A (en) * 1984-08-24 1988-05-17 University Patents, Inc. Herpes virus specific immunological materials and methods
US4658022A (en) * 1985-08-08 1987-04-14 Molecular Diagnostics, Inc. Binding of antibody reagents to denatured protein analytes
US4959323A (en) * 1985-11-04 1990-09-25 Mt. Sinai School Of Medicine Of The City University Of New York Production and use of pre S polypeptides of hepatitis B virus
US4904581A (en) * 1986-05-06 1990-02-27 Epitope, Inc. Method of detecting AIDS virus infection
US5106726A (en) * 1990-02-16 1992-04-21 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV

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DK0529057T3 (da) 1997-08-25
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WO1992015881A1 (fr) 1992-09-17
DE69220863T2 (de) 1998-02-26
CA2081657A1 (fr) 1992-09-07
ES2106179T3 (es) 1997-11-01
ATE155583T1 (de) 1997-08-15
AU1642792A (en) 1992-10-06
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JP3307637B2 (ja) 2002-07-24
EP0529057A4 (en) 1993-10-20
AU646738B2 (en) 1994-03-03
DE69220863D1 (de) 1997-08-21
CA2081657C (fr) 2003-10-14

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